Numerous groundwater aquifers are contaminated with organic and inorganic pollutants. Directly monitoring biodegrading microbial communities could guide decisions about treatment and facilitate monitoring of remediation. A common, but not well-investigated, challenge at Superfund sites is managing remediation of mixtures of contaminants. Superfund chemicals, toluene, benzene and xylene, along with fuel oxygenates, comprise mixed contaminants associated with petroleum pollution. Our aim is to adapt and develop molecular tools to assess the behavior of microbial communities associated with mixtures of pollutants. The underlying hypothesis that will guide our technology development is that microbial community structure (diversity and numbers of total bacteria, certain anaerobic toluene/xylene degraders, and denitrifiers) and community functions (e.g. rates of utilization of pollutants as e donors, use of electron acceptors) when exposed to mixtures of contaminants will not behave as predicted from behavior on single contaminants. We will focus on toluene and xylene biodegradation under nitrate reducing conditions, but also consider interactions of these processes with other BTEX compounds and the fuel additives, methyl tertiary butyl ether (MTBE) and ethanol. Our study system will be groundwater aquifer microbial communities, in microcosm and controlled field studies. Questions to be addressed include: 1) Are differences in biodegradation rates of contaminants, alone versus mixed with other chemicals, associated with differences in diversity and numbers of total bacteria, toluene/xylene degraders, and denitrifiers? 2) Do the same microbial populations use multiple contaminants? 3) Do readily degradable (e.g., ethanol) and recalcitrant (e.g., MTBE) fuel additives alter the biodegradation of BTEX under anaerobic conditions? We will use DGGE analysis and real-time quantitative PCR, targeting primers for enzymes involved in contaminant degradation (bssA) and nitrate reduction (nirS and nirK) and total bacterial cells (universal bacterial 16S rDNA). We will measure the accumulation of carbon from 14C labeled compounds (toluene, MTBE) into cellular constituents (PLFA, determine signature lipids/markers) of microbial communities using GC-MS and AMS (at Lawrence Livermore National Lab) in artificial mixes of bacterial strains and in microcosms. We will conduct studies in microcosms and across pollutant gradients (of BTEX, BTEX + MTBE or ethanol) at a field site containing a petroleum spill (with BTEX and MTBE) at Vandenberg Air Force Base in CA.